Myelodysplastic syndromes (MDS) are generally incurable hematologic disorders associated with disease initiating stem cells that are not eliminated by conventional therapies and need to be targeted for potentially curative strategies. We recently demonstrated that aberrant hematopoietic stem cells are expanded in MDS, can persist during phenotypic remissions and can predict relapse. STAT3 is a transcription factor that was found to be overexpressed in MDS stem cells and higher expression was associated with an adverse prognosis in a large cohort of patients. We also obtained proof-of-concept data that inhibition of STAT3 can target malignant stem cells while sparing healthy control stem and progenitors, thus identifying it as a potential therapeutic target in MDS. To comprehensively examine the role of this pathway in MDS, Aim 1 will define the functional role of STAT3 on growth of disease initiating stem cells in MDS and determine the efficacy of clinically relevant inhibitors of this pathway in large cohort of primary human samples. Additionally, in vitro and in vivo efficacy of a clinically applicable, anti-sense inhibitor (AZD-9150) against a large number of primary MDS samples will be correlated with clinical and mutational subtypes to identify subsets that will be sensitive to STAT3 inhibition. Patient derived MDS xenografts will also be used to determine in vivo efficacy. Aim 2 will determine the requirement for STAT3 in initiation of dysplasia/disease progression in vivo by genetic deletion of STAT3 in two mouse models of MDS. Along with the NUP-HOXD13 model; a novel model of MDS dysplasia and transformation which we have recently developed, induced by heterozygous PU.1 enhancer deletion, will be used to study the effect of STAT3 deletion on disease initiating stem cells and disease progression. Aim 3 will identify the downstream effectors of STAT3 activation in MDS. Combination of Chip-seq and transcriptomic profiling will be used to identify direct targets of STAT3 activation, that will tested in functional assays in MDS models. We will also determine the role of MCL-1 as a downstream anti-apoptotic target of STAT3 activation in MDS stem cells. Identification of critical downstream effectors will improve our molecular understanding of the STAT3 pathway in MDS and will be instrumental to develop more specific and potent strategies to inhibit this pathway. Taken together, these studies will study the role of the STAT3 pathway in MDS pathogenesis and determine its potential as a therapeutic target against immature, disease initiating cells in MDS.